Decrease In Cytosolic pH Research Articles

Salt-Induced Changes in Cytosolic pH and Photosynthesis in Tobacco and Potato Leaves.

Salinity is one of the most common factors limiting the productivity of crops. The damaging effect of salt stress on many vital plant processes is mediated, on the one hand, by the osmotic stress caused by large concentrations of Na+ and Cl- outside the root and, on the other hand, by the toxic effect of these ions loaded in the cell. In our work, the influence of salinity on the changes in photosynthesis, transpiration, water content and cytosolic pH in the leaves of two important crops of the Solanaceae family-tobacco and potato-was investigated. Salinity caused a decrease in photosynthesis activity, which manifested as a decrease in the quantum yield of photosystem II and an increase in non-photochemical quenching. Along with photosynthesis limitation, there was a slight reduction in the relative water content in the leaves and a decrease in transpiration, determined by the crop water stress index. Furthermore, a decrease in cytosolic pH was detected in tobacco and potato plants transformed by the gene of pH-sensitive protein Pt-GFP. The potential mechanisms of the salinity influence on the activity of photosynthesis were analyzed with the comparison of the parameters' dynamics, as well as the salt content in the leaves.

Mefloquine causes selective mast cell apoptosis in cutaneous mastocytosis lesions by a secretory granule-mediated pathway.

Mastocytosis is a KIT-related myeloproliferative disease characterised by abnormal expansion of neoplastic mast cells (MC) in the skin or virtually any other organ system. The cutaneous form of adult-onset mastocytosis is almost invariably combined with indolent systemic involvement for which curative therapy is yet not available. Here we evaluated a concept of depleting cutaneous MCs in mastocytosis lesions ex vivo by targeting their secretory granules. Skin biopsies from mastocytosis patients were incubated with or without mefloquine, an antimalarial drug known to penetrate into acidic organelles such as MC secretory granules. Mefloquine reduced the number of dermal MCs without affecting keratinocyte proliferation or epidermal gross morphology at drug concentrations up to 40 μM. Flow cytometric analysis of purified dermal MCs showed that mefloquine-induced cell death was mainly due to apoptosis and accompanied by caspase-3 activation. However, caspase inhibition provided only partial protection against mefloquine-induced cell death, indicating predominantly caspase-independent apoptosis. Further assessments revealed that mefloquine caused an elevation of granule pH and a corresponding decrease in cytosolic pH, suggesting drug-induced granule permeabilisation. Extensive damage to the MC secretory granules was confirmed by transmission electron microscopy analysis. Further, blockade of granule acidification or serine protease activity prior to mefloquine treatment protected MCs from apoptosis, indicating that granule acidity and granule-localised serine proteases play major roles in the execution of mefloquine-induced cell death. Altogether, these findings reveal that mefloquine induces selective apoptosis of MCs by targeting their secretory granules and suggest that the drug may potentially extend its range of medical applications.

Human lactate dehydrogenase A undergoes allosteric transitions under pH conditions inducing the dissociation of the tetrameric enzyme.

The aerobic energetic metabolism of eukaryotic cells relies on the glycolytic generation of pyruvate, which is subsequently channelled to the oxidative phosphorylation taking place in mitochondria. However, under conditions limiting oxidative phosphorylation, pyruvate is coupled to alternative energetic pathways, e.g. its reduction to lactate catalyzed by lactate dehydrogenases (LDHs). This biochemical process is known to induce a significant decrease in cytosolic pH, and is accordingly denoted lactic acidosis. Nevertheless, the mutual dependence of LDHs action and lactic acidosis is far from being fully understood. Using human LDH-A, here we show that when exposed to acidic pH this enzyme is subjected to homotropic allosteric transitions triggered by pyruvate. Conversely, human LDH-A features Michaelis–Menten kinetics at pH values equal to 7.0 or higher. Further, citrate, isocitrate, and malate were observed to activate human LDH-A, both at pH 5.0 and 6.5, with citrate and isocitrate being responsible for major effects. Dynamic light scattering (DLS) experiments revealed that the occurrence of allosteric kinetics in human LDH-A is mirrored by a consistent dissociation of the enzyme tetramer, suggesting that pyruvate promotes tetramer association under acidic conditions. Finally, using the human liver cancer cell line HepG2 we isolated cells featuring cytosolic pH equal to 7.3 or 6.5, and we observed a concomitant decrease in cytosolic pH and lactate secretion. Overall, our observations indicate the occurrence of a negative feedback between lactic acidosis and human LDH-A activity, and a complex regulation of this feedback by pyruvate and by some intermediates of the Krebs cycle.

Extracellular acidification induces ROS- and mPTP-mediated death in HEK293 cells

The extracellular pH (pHe) is a key determinant of the cellular (micro)environment and needs to be maintained within strict boundaries to allow normal cell function. Here we used HEK293 cells to study the effects of pHe acidification (24h), induced by mitochondrial inhibitors (rotenone, antimycin A) and/or extracellular HCl addition. Lowering pHe from 7.2 to 5.8 reduced cell viability by 70% and was paralleled by a decrease in cytosolic pH (pHc), hyperpolarization of the mitochondrial membrane potential (Δψ), increased levels of hydroethidine-oxidizing ROS and stimulation of protein carbonylation. Co-treatment with the antioxidant α-tocopherol, the mitochondrial permeability transition pore (mPTP) desensitizer cyclosporin A and Necrostatin-1, a combined inhibitor of Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and Indoleamine 2,3-dioxygenase (IDO), prevented acidification-induced cell death. In contrast, the caspase inhibitor zVAD.fmk and the ferroptosis inhibitor Ferrostatin-1 were ineffective. We conclude that extracellular acidification induces necroptotic cell death in HEK293 cells and that the latter involves intracellular acidification, mitochondrial functional impairment, increased ROS levels, mPTP opening and protein carbonylation. These findings suggest that acidosis of the extracellular environment (as observed in mitochondrial disorders, ischemia, acute inflammation and cancer) can induce cell death via a ROS- and mPTP opening-mediated pathogenic mechanism.

TM9SF4 is a novel V-ATPase-interacting protein that modulates tumor pH alterations associated with drug resistance and invasiveness of colon cancer cells.

An inverted pH gradient across the cell membranes is a typical feature of malignant cancer cells that are characterized by extracellular acidosis and cytosol alkalization. These dysregulations are able to create a unique milieu that favors tumor progression, metastasis and chemo/immune-resistance traits of solid tumors. A key event mediating tumor cell pH alterations is an aberrant activation of ion channels and proton pumps such as (H+)-vacuolar-ATPase (V-ATPase). TM9SF4 is a poorly characterized transmembrane protein that we have recently shown to be related to cannibal behavior of metastatic melanoma cells. Here, we demonstrate that TM9SF4 represents a novel V-ATPase-associated protein involved in V-ATPase activation. We have observed in HCT116 and SW480 colon cancer cell lines that TM9SF4 interacts with the ATP6V1H subunit of the V-ATPase V1 sector. Suppression of TM9SF4 with small interfering RNAs strongly reduces assembly of V-ATPase V0/V1 sectors, thus reversing tumor pH gradient with a decrease of cytosolic pH, alkalization of intracellular vesicles and a reduction of extracellular acidity. Such effects are associated with a significant inhibition of the invasive behavior of colon cancer cells and with an increased sensitivity to the cytotoxic effects of 5-fluorouracil. Our study shows for the first time the important role of TM9SF4 in the aberrant constitutive activation of the V-ATPase, and the development of a malignant phenotype, supporting the potential use of TM9SF4 as a target for future anticancer therapies.

Talin regulates moesin-NHE-1 recruitment to invadopodia and promotes mammary tumor metastasis.

Invadopodia are actin-rich protrusions that degrade the extracellular matrix and are required for stromal invasion, intravasation, and metastasis. The role of the focal adhesion protein talin in regulating these structures is not known. Here, we demonstrate that talin is required for invadopodial matrix degradation and three-dimensional extracellular matrix invasion in metastatic breast cancer cells. The sodium/hydrogen exchanger 1 (NHE-1) is linked to the cytoskeleton by ezrin/radixin/moesin family proteins and is known to regulate invadopodium-mediated matrix degradation. We show that the talin C terminus binds directly to the moesin band 4.1 ERM (FERM) domain to recruit a moesin-NHE-1 complex to invadopodia. Silencing talin resulted in a decrease in cytosolic pH at invadopodia and blocked cofilin-dependent actin polymerization, leading to impaired invadopodium stability and matrix degradation. Furthermore, talin is required for mammary tumor cell motility, intravasation, and spontaneous lung metastasis in vivo. Thus, our findings provide a novel understanding of how intracellular pH is regulated and a molecular mechanism by which talin enhances tumor cell invasion and metastasis.

CO2 Transport by PIP2 Aquaporins of Barley

CO2 permeability of plasma membrane intrinsic protein 2 (PIP2) aquaporins of Hordeum vulgare L. was investigated. Five PIP2 members were heterologously expressed in Xenopus laevis oocytes. CO2 permeability was determined by decrease of cytosolic pH in CO2-enriched buffer using a hydrogen ion-selective microelectrode. HvPIP2;1, HvPIP2;2, HvPIP2;3 and HvPIP2;5 facilitated CO2 transport across the oocyte cell membrane. However, HvPIP2;4 that is highly homologous to HvPIP2;3 did not. The isoleucine residue at position 254 of HvPIP2;3 was conserved in PIP2 aquaporins of barley, except HvPIP2;4, which possesses methionine instead. CO2 permeability was lost by the substitution of the Ile254 of HvPIP2;3 by methionine, while water permeability was not affected. These results suggest that PIP2 aquaporins are permeable to CO2. and the conserved isoleucine at the end of the E-loop is crucial for CO2 selectivity.

Possible causes of apoptotic volume decrease: an attempt at quantitative review

Cell shrinkage and dehydration are essential characteristics of apoptosis, and loss of as much as half of the initial cell volume is not uncommon. This phenomenon is usually explained by efflux of K(+) and Cl(-). We reexamine this hypothesis on the basis of the available data for ion concentrations and the requirements for osmotic equilibrium and electroneutrality. In addition to ion loss, we discuss the possible impacts of several other processes: efflux of low-molecular-weight osmolytes, acidification of the cytosol, effects of water channels and pumps, heterogeneity of intracellular water, and dissociation of apoptotic bodies. We conclude that most mammalian cells are theoretically capable of reducing their volume by 15-20% through ion loss or a decrease in cytosolic pH, although, in reality, the contribution of these mechanisms to apoptotic shrinkage may be smaller. Transitions between osmotically active and inactive water pools might influence cell volume as well; these mechanisms are poorly understood but are amenable to experimental study. Dissociation of apoptotic bodies is a separate mechanism of volume reduction and should be monitored closely; this can be best achieved by measurement of intracellular water, rather than cell volume.

The Arabidopsis central vacuole as an expression system for intracellular transporters: functional characterization of the Cl−/H+ exchanger CLC‐7

Functional characterization of intracellular transporters is hampered by the inaccessibility of animal endomembranes to standard electrophysiological techniques. Here, we used Arabidopsis mesophyll protoplasts as a novel heterologous expression system for the lysosomal chloride–proton exchanger CLC-7 from rat. Following transient expression of a rCLC-7:EGFP construct in isolated protoplasts, the fusion protein efficiently targeted to the membrane of the large central vacuole, the lytic compartment of plant cells. Membrane currents recorded from EGFP-positive vacuoles were almost voltage independent and showed time-dependent activation at elevated positive membrane potentials as a hallmark. The shift in the reversal potential of the current induced by a decrease of cytosolic pH was compatible with a 2Cl(-)/1H(+) exchange stoichiometry. Mutating the so-called gating glutamate into alanine (E245A) uncoupled chloride fluxes from the movement of protons, transforming the transporter into a chloride channel-like protein. Importantly, CLC-7 transport activity in the vacuolar expression system was recorded in the absence of the auxiliary subunit Ostm1, differently to recent data obtained in Xenopus oocytes using a CLC-7 mutant with partial plasma membrane expression. We also show that plasma membrane-targeted CLC-7(E245A) is non-functional in Xenopus oocytes when expressed without Ostm1. In summary, our data suggest the existence of an alternative CLC-7 operating mode, which is active when the protein is not in complex with Ostm1. The vacuolar expression system has the potential to become a valuable tool for functional studies on intracellular ion channels and transporters from animal cells.

Unique Dimeric Structure of BNip3 Transmembrane Domain Suggests Membrane Permeabilization as a Cell Death Trigger

BNip3 is a prominent representative of apoptotic Bcl-2 proteins with rather unique properties initiating an atypical programmed cell death pathway resembling both necrosis and apoptosis. Many Bcl-2 family proteins modulate the permeability state of the outer mitochondrial membrane by forming homo- and hetero-oligomers. The structure and dynamics of the homodimeric transmembrane domain of BNip3 were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics energy relaxation in an explicit lipid bilayer. The right-handed parallel helix-helix structure of the domain with a hydrogen bond-rich His-Ser node in the middle of the membrane, accessibility of the node for water, and continuous hydrophilic track across the membrane suggest that the domain can provide an ion-conducting pathway through the membrane. Incorporation of the BNip3 transmembrane domain into an artificial lipid bilayer resulted in pH-dependent conductivity increase. A possible biological implication of the findings in relation to triggering necrosis-like cell death by BNip3 is discussed.

Free Mg2+ concentration in the calf muscle of glycogen phosphorylase and phosphofructokinase deficiency patients assessed in different metabolic conditions by 31P MRS.

BackgroundThe increase in cytosolic free Mg2+ occurring during exercise and initial recovery in human skeletal muscle is matched by a decrease in cytosolic pH as shown by in vivo phosphorus magnetic resonance spectroscopy (31P MRS). To investigate in vivo to what extent the homeostasis of intracellular free Mg2+ is linked to pH in human skeletal muscle, we studied patients with metabolic myopathies due to different disorders of glycogen metabolism that share a lack of intracellular acidification during muscle exercise.MethodsWe assessed by 31P MRS the cytosolic pH and free magnesium concentration ([Mg2+]) in calf muscle during exercise and post-exercise recovery in two patients with McArdle's disease with muscle glycogen phosphorylase deficiency (McArdle), and two brothers both affected by Tarui's disease with muscle phosphofructokinase deficiency (PFK).ResultsAll patients displayed a lack of intracellular acidosis during muscle exercise. At rest only one PFK patient showed a [Mg2+] higher than the value found in control subjects. During exercise and recovery the McArdle patients did not show any significant change in free [Mg2+], while both PFK patients showed decreased free [Mg2+] and a remarkable accumulation of phosphomonoesters (PME). During initial recovery both McArdle patients showed a small increase in free [Mg2+] while in PFK patients the pattern of free [Mg2+] was related to the rate of PME recovery.Conclusioni) homeostasis of free [Mg2+] in human skeletal muscle is strongly linked to pH as shown by patients' [Mg2+] pattern during exercise;ii) the pattern of [Mg2+] during exercise and post-exercise recovery in both PFK patients suggests that [Mg2+] is influenced by the accumulation of the phosphorylated monosaccharide intermediates of glycogenolysis, as shown by the increased PME peak signal.iii) 31P MRS is a suitable tool for the in vivo assessment of free cytosolic [Mg2+] in human skeletal muscle in different metabolic conditions;

Ortho-substituted PCB95 alters intracellular calcium signaling and causes cellular acidification in PC12 cells by an immunophilin-dependent mechanism.

ortho-Substituted PCBs mobilize Ca2+ from isolated brain microsomes by interaction with FKBP12/RyR complexes. Investigation into the cellular importance of this mechanism was undertaken using PC12 cells by fluoroimaging the actions of specific PCB congeners on [Ca2+]i and pH. RyR and IP3R share a common intracellular Ca2+ store in PC12 cells. Perfusion of nM to low microM PCB95 caused a transient rise of [Ca2+]i that was not completely dependent on extracellular Ca2+. Pre-incubation of the cells with ryanodine or FK506 completely eliminated PCB95 responses, suggesting a primary action on the FKPP12/RyR-sensitive store. PCB95, but not PCB126, induced a gradual decrease in cytosolic pH that could be completely eliminated by FK506 pre-incubation of the cells. Direct respiration measurement using isolated brain mitochondria demonstrated that neither of the PCBs directly altered any stage of mitochondrial respiration. These results revealed that PCB95 disrupts intracellular Ca2+ signaling in PC12 cells by interaction with the FKBP12/RyR complex that in turn accelerated cellular metabolism, possibly affecting signaling between ER and mitochondria. Since ortho-substituted PCBs have been shown to be neurotoxic and may affect neurodevelopment, studies on the molecular mechanism by which they alter cellular signaling may provide valuable information on the physiological roles of FKPB12 and RyR on neuronal functions.

The H(+)-ATPase in the plasma membrane of Saccharomyces cerevisiae is activated during growth latency in octanoic acid-supplemented medium accompanying the decrease in intracellular pH and cell viability.

Saccharomyces cerevisiae plasma membrane H(+)-ATPase activity was stimulated during octanoic acid-induced latency, reaching maximal values at the early stages of exponential growth. The time-dependent pattern of ATPase activation correlated with the decrease of cytosolic pH (pHi). The cell population used as inoculum exhibited a significant heterogeneity of pHi, and the fall of pHi correlated with the loss of cell viability as determined by plate counts. When exponential growth started, only a fraction of the initial population was still viable, consistent with the role of the physiology and number of viable cells in the inoculum in the duration of latency under acid stress.

RNase Activities Are Reduced Concomitantly with Conservation of Total Cellular RNA and Ribosomes in O2-Deprived Seedling Roots of Maize.

The effect of O2 deprivation on the activities of RNases and levels of total cellular RNA and ribosomes in seedling roots of maize (Zea mays L.) was investigated. Sodium dodecyl sulfate-polyacrylamide gels containing RNA were used to distinguish RNase isoenzymes by apparent molecular mass. Since O2 deprivation causes a decrease in cytosolic pH from approximately pH 7.4 to 6.4 and an elevation in cytosolic Ca2+, RNase levels were examined in the physiological range of cytosolic pH and in the presence of Ca2+, Mg2+, Zn2+, ethylenediaminetetracetate, or ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime]-tetraacetic acid. The activity of a number of RNases present in aerobic roots was reduced in response to O2 deprivation. Several RNases with a pH optimum of 6.4 were rapidly down-regulated by O2 deprivation. Spectrophotometric assay of extracts revealed that RNase activity was higher at pH 6.4 than at 7.2, and ethylenediaminetetracetate-insensitive RNase activity decreased in response to O2 deprivation. The decrease in RNase activity was correlated with no loss of total cellular RNA or ribosomes, despite a 4-fold decrease in run-on transcription of rRNA in isolated nuclei. Regulation of RNase activity may facilitate the conservation of nontranslating ribosomes and poorly translated mRNAs during O2 deprivation.

Effect of extracellular acidification on the activity of plasma membrane ATPase and on the cytosolic and vacuolar pH of Saccharomyces cerevisiae

The rapid in vivo activation of Saccharomyces cerevisiae plasma membrane H +-ATPase that has been attributed to medium acidification from pH 6.5 to pH 3.5 is not caused by the low pH itself but is induced by the weak organic acid (succinic) used as the acidulant. The activation induced by 50 mM succinic acid at pH 3.5 occurred in both the presence or absence of glucose. Activation at pH 3.5 was also induced by acetic acid and it was maximal at 50 mM concentration. To investigate the role of plasma membrane ATPase activation in pH homeostasis, the internal pH (cytosolic and vacuolar) of yeast cells incubated in media at pH 6.5 or at pH 3.5, acidified either with HCl or acetic acid, were compared by using in vivo 31P-NMR. Despite plasma membrane ATPase activation by acetic acid, the decrease in cytosolic pH caused by external acidification was much more important when the permeant acetic acid was used instead of HCl as the acidulant. The supplementation of the incubation medium at pH 3.5 with glucose led to higher cytosolic pH values, consistent with the observed in vivo activation of plasma membrane ATPase by glucose. At the external pH value of 6.5 the vacuole was maintained at a mildly acidic pH (around 6) while the cytosol was at about neutral pH; however, when cytoplasmic pH decreased due to external acidification, vacuolar pH accompanied that decrease. Vacuolar pH reached 5.4–5.5 during incubation with HCl and dropped sharply to values below 4.4 in cells incubated with acetic acid. These results indicate that the vacuole also plays a role in homeostasis of the intracellular pH.

Purification and Structural and Kinetic Characterization of the Pyrophosphate:Fructose-6-Phosphate 1-Phosphotransferase from the Crassulacean Acid Metabolism Plant, Pineapple.

Pyrphosphate-dependent phosphofructokinase (PFP) was purified to electrophoretic homogeneity from illuminated pineapple (Ananas comosus) leaves. The purified enzyme consists of a single subunit of 61.5 kD that is immunologically related to the potato tuber PFP [beta] subunit. The native form of PFP likely consists of a homodimer of 97.2 kD, as determined by gel filtration. PFP's glycolytic activity was strongly dependent on pH, displaying a maximum at pH 7.7 to 7.9. Gluconeogenic activity was relatively constant between pH 6.7 and 8.7. Activation by Fru-2,6-bisphosphate (Fru-2,6-P2) was dependent on assay pH. In the glycolytic direction, it activated about 10-fold at pH 6.7, but only 2-fold at pH 7.7. The gluconeogenic reaction was only weakly affected by Fru-2,6-P2. The true substrates for the PFP forward and reverse reactions were Fru-6-phosphate and Mg-pyrophosphate, and Fru-1,6-P2, orthophosphate, and Mg2+, respectively. The results suggest that pineapple PFP displays regulatory properties consistent with a pH-based regulation of its glycolytic activity, in which a decrease in cytosolic pH caused by nocturnal acidification during Crassulacean acid metabolism, which could curtail its activity, is compensated by a parallel increase in its sensitivity to Fru-2,6-P2. It is also evident that the [beta] subunit alone is sufficient to confer PFP with a high catalytic rate and the regulatory properties associated with activation by Fru-2,6-P2.

Palytoxin modulates cytosolic pH in human osteoblast-like Saos-2 cells via an interaction with Na(+)-K(+)-ATPase.

Palytoxin (PTx) at nanomolar concentrations enhances the permeability of mammalian cell membranes to both Na+ and Ca2+. In basal human osteoblast-like Saos-2 cells, PTx (8 nM) caused a persistent decrease in cytosolic pH (pHi) of about 0.2 units, which required the presence of extracellular Ca2+ (Cae2+) and Na+ (Nae+). We acidified Saos-2 cells by incubation with nigericin to examine the action of PTx in cells with an activated Na+/H+ antiporter. Under these conditions, PTx increased the pHi without requiring Cae2+ or Nae+, and the alkalinization was unaffected by hexamethylene amiloride. We conclude that the PTx-induced rise in pHi did not involve the Na+/H+ antiporter. PTx increased the rate of 86Rb+ efflux. We propose that PTx induced alkalinization in nigericin-acidified cells by collapsing the K+ gradient. Exposure to ouabain had no effect on pHi, but it prevented the actions of PTx on PHi in both basal and nigericin-acidified cells. Ouabain-resistant mutant cells were less sensitive to PTx in extruding 86Rb+ than their ouabain-sensitive parents. We conclude that PTx interacts with the Na(+)-K(+)-adenosinetriphosphatase to regulate pHi in both basal and nigericin-acidified Saos-2 cells.

Carrier-mediated transport of lactic acid in cultured neurons and astrocytes.

The glycolytic end product lactic acid induced a rapid transient decrease in cytosolic pH in cultured neurons and astrocytes, as measured by microspectrofluorometry using the fluorescent indicator dye 2',7'-bis-(2-carboxyethyl)-5-(and-6) carboxyfluorescein acetoxymethyl ester. Over a physiological range of pH, the initial rate of cellular acidification was a saturable function of the extracellular lactate concentration, suggesting that a saturable transport system mediated lactic acid permeation across the plasma membrane. This transport process displayed stereoselectivity, with a threefold higher rate of intracellular acidification by L-lactic acid than by its D-isomer. Lactic acid-induced acidification occurred in the absence of intracellular ATP, suggesting that transport proceeded independently of the cellular energy charge. These data suggest the existence of a lactic acid carrier in mammalian neuronal and astrocytic plasma membranes, which might serve an acid-scavenging function under conditions of altered pH homeostasis. In the setting of in vivo cerebral ischemia, this carrier may promote the efflux of lactic acid from astrocytes, redistributing it among less metabolically active neurons.

Relaxing Effect of Vesnarinone (OPC-8212) on the Tracheal Muscle Strips Isolated from Guinea Pigs

A transient decrease in cytosolic pH ([pH]i) in rat parotid cells was evoked by the addition of carbachol (CCh), phenylephrine, or substance P, whereas isoproterenol and dibutyryl cyclic AMP had little or no effect on [pH]i. The decrease in [pH]i induced by the Ca(2+)-mobilizing agonists was also observed in Ca(2+)-free medium, but not when the intracellular Ca2+ stores were previously depleted. Ionomycin and thapsigargin elicited a decrease in [pH]i with an increase in cytosolic Ca2+ concentration ([Ca2+]i). The protein kinase C activator and inhibitor had no effect on the agonist-induced decrease in [pH]i. These results suggest that the cytosolic acidification is associated with an increase in [Ca2+]i.

In vivo and in vitro 31P-NMR spectroscopy of rat liver treated with halocarbons

Both in vivo and in vitro 31P-NMR spectroscopy were used to demonstrate metabolic changes in rat liver as a function of time after exposure to either carbon tetrachloride (CCl 4) or bromotrichloromethane (BrCCl 3). The inorganic phosphate resonance, measured in vivo, moves upfield, which is associated with a decrease in cytosolic pH over a 12 or 20 h period (for BrCCl 3 or CCl 4, respectively). Intoxication by CCl 4 or BrCCl 3 causes an intracellular acidosis to pH 7.05 or 6.82 (±0.05), respectively. Also, it has been found that halocarbon exposure increases the amounts of phosphomonoesters (PME) detected. High resolution in vitro 31P-NMR spectroscopy studies of perchloric acid extracts of CCl 4-treated rat livers indicated a significant increase in the height of the phosphocholine resonance in the PME region 4–5 h after CCl 4 exposure.